RNA interference (RNAi) has become a widely used tool for functional genomic studies in vertebrate and invertebrates. RNAi works by silencing a gene through homologous short interfering dsRNAs (for example siRNAs), which trigger the destruction of the corresponding mRNA by the RNA-induced silencing complex (RISC). The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms. Synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. The effect of these genes on the phenotype of the cells can then be analyzed by studying the effect of the gene silencing. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as for example, cell division.
Due to its advantages, in particular siRNA-mediated RNAi has become an indispensable tool in functional genomic research. Chemically synthesized siRNA reagents that target every gene in a human, mouse and rat genome are available for convenient delivery in vitro. Data acquired from RNAi experiments are used to support important conclusions about how genes function. For the above reasons, the RNA interference pathway is often exploited in experimental biology to study the function of genes in cell culture and in vivo in model organisms. Double-stranded RNA is synthesized with a sequence complementary to the target sequence of a gene of interest, usually an 18 to 30 mer and introduced into the cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers can cause a drastic decrease in the expression of the targeted gene. Studying the effects of this decrease can show the physiological role of the respective targeted gene product. Since RNAi may not necessarily totally abolish expression of the gene, this technique is sometimes referred to as a “knockdown” to distinguish it from “knockout” procedures, in which expression of a gene is entirely eliminated, e.g. by introducing a knock-out mutation in the target gene and thus the DNA. The ease, speed, and cost-effectiveness have made RNAi the method of choice in particular for loss-of-gene function studies.
However, even though RNAi is a valuable tool for several applications in research as well as in therapeutic applications it has produced a new set of problems regarding the specificity of the RNAi mediating molecule. It was shown that unspecific off-target effects of the transfected/provided RNAi mediating compounds may constitute a major problem. This, as the off-target effects may overlay the specific effect of the transfected RNAi mediating compound (e.g. siRNA). Many off-target effects of transfected RNAi inducing compounds such as siRNA occur because the siRNA may act like a miRNA, by binding to partially homologue sequences in the 3′ UTR region of the mRNAs. This binding may lead to an unwanted regulation of mRNA targets, which are not supposed to be targeted/regulated by the particular RNAi mediating compound.
Although it is possible to achieve single base discrimination with selected siNAs, single—or even double—base mismatches are often tolerated and can still reduce target levels by significant amounts. If the full sequence of the reference genome is known, a thorough homology screen of candidate siNAs might permit exclusion of sites where unwanted homology exists with other genes and theoretically lead to high specificity. Unfortunately, this kind of traditional cross-hybridization analysis can be less effective than expected and even carefully screened siNAs can cause significant changes in the expression level of unrelated genes. As it is outlined above, it appears that many of these effects are mediated by the unintended participation of siNAs in miRNA pathways.
The miRNA translational suppression pathway is often directed by imperfect base pairing between the target and antisense strand and the specificity of this process is defined by 6 to 8 base “seed region” at the 5′ end of the antisense strand of the miRNA. Given the expected frequency of finding 6 to 7 base matches between a siNA and a non target gene within the entire transcriptome, it is not surprising that off-target effects mediated by this mechanism can simultaneously affect hundreds of genes. Target sites for miRNA binding seem to be enriched in the 3′-untranslated region (3′UTR) of genes and a careful focus on homology screening of the seed regions of candidate siRNAs in the 3′UTR of all genes may be prudent. Unfortunately, 6-base matches are very common, and only a few of these matches actually proved to be real functional sites that can lead to target gene suppression.
Therefore, means for reducing in particular miRNA pathway derived off-target effects is needed beyond seed region homology screening and proper controls and comparative experiments in order to obtain reliable RNAi based results in order to enhance the specificity e.g. for research or therapeutic applications of the siNA.
Although siNAs such as in particular siRNAs have two strands, only functional participation of the guide strand (also referred to as the antisense strand) is desired. Design of the siNA can introduce strand bias into the siNA so that one strand is preferentially incorporated into RISC. However, this bias is only relative and some variable amount of the passenger strand (also referred to as the sense strand) will be functionally loaded and can be engaged in undesired gene knock-down events. Various strategies has been developed in the prior art to reduce or totally eliminate participation of the sense strand of the siNA in gene silencing. One approach is to cleave the sense strand so that the RNA duplex is comprised of an intact antisense strand which is annealed to two adjoining shorter sense strand RNA oligomers. This design has been called “small internally segmented siRNA”. Another approach is to use modifications that block the 5′ end of the sense strand of the siNA. Both strands of the endogenous siRNAs or miRNAs naturally have a 5′-phosphate (which results from Dicer cleavage). Synthetic siNAs are usually made with a 5′-phosphate or 5′-hydroxyl (in which case the siRNAs phosphorylated by the cellular RNA kinase hClp1). Although synthetic siNAs are tolerant of some 5′-modifications, blocking the 5′end of a siNA strand (by, for example, 5′-O-methylation) can reduce or eliminate participation of that strand in silencing.
The use of site-specific chemical modification may also permit reduction or elimination of off-target effects derived from unwanted participation of the antisense strand in miRNA pathways. Several groups have studied chemical modification patterns looking for selective modification strategies that retain potency of the siNA antisense strand to direct Ago2 cleavage of an mRNA target while reducing participation of that siNA in miRNA-like seed-region directed events. For example placing a single 2′OMe residue at position +2 of the guide strand can substantially reduce seed region-related off-target effects. Furthermore, replacing the entire seed region with DNA residues maintained functional potency for knockdown of the intended target while reducing seed region-related off-target effects.
Thus, the use of chemical modifications in the siNA molecules may reduce off-target effects that arise either from triggering the innate immune system as well as reduce the ability of a siNA to participate in seed-region directed miRNA-pathway off-target effects.
It is the object of the present invention to provide a siNA molecule which is capable of efficiently down regulating a target gene via RNA interference which shows reduced off-target effects, in particular compared to unmodified molecules. In particular, the unwanted participation of the antisense strand in miRNA pathways is to be reduced.